Correlation between antibiotic resistance and phylogenetic types among multidrug-resistant Escherichia coli isolated from urinary tract infections

Objective(s): Emergence of multidrug resistance has reduced the choice of antimicrobial regimens for UTIs. To understand the association of phenotype and genotype among uropathogens. Materials and Methods: Six hundred and twenty-eight (628) urine samples were collected and analyzed. Antibiotic sensitivity pattern was determined by the Kirby-Bauer Disc Diffusion Method and minimum inhibitory concentration (MIC) was tested by the E test. Fluoroquinolone resistant mutations in QRDR of gyrA and ParC, phylogenetic groups, and PAIusp subtype were detected by PCR. Results: Most prevalent uropathogens were Escherichia coli (53.2%) followed by Klebsiella pneumoniae (21%). Multidrug- resistance was observed in > 50% cases for third-generation cephalosporins and ciprofloxacin and lowest in meropenem. E. coli (66.2%) and K. pneumonia (64.4%) were extended-spectrum β-lactamases (ESBLs) producers. MIC to trimethoprim-sulfamethoxazole was highest in E. coli (>1024 µg/ml). In 80 (24%) of the 334 E. coli isolates analyzed in detail, 54 fluoroquinolones (FQ) resistant isolates carried mutations (S83L, D87N, S80I, E84V) in QRDR of gyrA and ParC. Out of 54 FQ-resistant isolates, 43 (79.6%) isolates belonged to the phylogenetic group B2, and 11(20.4%) belonged to group D. Isolates belonged to group B2, 38 (88.4%) of the 43 isolates carried PAIusp subtype IIa and high frequency of mutation E84V in ParC was detected in 37 (97.4%). Other mutations, such as S80I, S83L in gyrA and D87N in ParC were found in all resistant isolates. Conclusion: Correlations between phenotype and genotype provided a basis to understand the resistance development in uropathogens, and PAIusp subtyping indicated that E. coli belonged to the B2 group.


Introduction
Urinary tract infection (UTI) is among the most common infectious diseases in both community and hospital settings. UTIs are caused by multidrugresistant bacteria and serves as a therapeutic challenge for general family practitioners and hospital doctors in developing countries (1,2). UTIs, if diagnosed early, and with adequate antibiotic coverage, are not alarming. However, if inadequately treated, these infections can cause significant morbidity and mortality (1,2). UTIs can be classified as community-acquired (CA) or hospitalacquired (HA). About 70 to 80% of CA-UTI is caused by multidrug-resistant Escherichia coli from the patient's normal gut flora. E. coli isolates that cause UTI and may cause extra-intestinal infections are responsible for epidemic spread within the population (3). In several studies, Gram-negative bacteria are responsible for 80-85% of UTIs and major causative organisms are E. coli (75-87%) followed by Klebsiella spp. Citrobacter spp, Enterobacter spp, Pseudomonas and Proteus spp (4,5). Women are more prone to UTIs than males. Higher incidence in females is due to urothelial mucosa adherence to mucopolysaccharide lining (6). In old age, the chance of UTI increases due to menopause (estrogen loss) in females. Furthermore, prolonged use of antibiotics can also damage periureteral flora resulting in more colonization of uropathogens to the urinary tract (7). UTIs are commonly treatable with antibiotics and the selection of empirical therapy should be based on the prevalent antibiogram data (8). The irrational use of antibiotics without proper antibiotic susceptibility testing has led to an extensive increase in drug-resistant UTIs pathogens that have made treatment more difficult. Overall, this situation has led to adverse consequences like high morbidity and mortality, prolonged hospital stays with higher costs and increased antimicrobial resistance, and a surge in nosocomial infections (9)(10)(11). The most commonly used antibiotics for routine UTIs are ß-lactam (penicillins, cephalosporins, and carbapenem), followed by fluoroquinolones, trimethoprim, trimethoprim/sulfamethoxazole, fosfomycin, nitrofurans and aminoglycosides (12). Fluoroquinolones are widely prescribed as empirical therapy in Europe and the USA (11,13). Acquisition of high-level resistance involves several factors and in the majority of cases, resistance arises spontaneously due to point mutation within gyrA, gyrB, parC, and pares genes (14). Qnr genes produce proteins that can bind to DNA gyrase, protecting it from the action of quinolones. Ultimately, the drugs effectively can be reduced with mutations at main sites in DNA gyrase or Topoisomerase IV, which can cause a decrease in their binding affinity to quinolones (15)(16)(17). Resistance to ß-lactam antibiotics among clinical isolates of E. coli and K. pneumoniae is most often due to the production of ß-lactamases (18,19). These enzymes confer resistance to extended-spectrum (third-generation) cephalosporins and monobactams (e.g., aztreonam). ESBLs genes are located on large ESBL plasmids which also confer resistance to other antibiotics including aminoglycosides, chloramphenicol, and sulfonamides and limiting their empirical and rational use for clinicians (20,21). Phylogenetic grouping of E. coli isolates is a more convenient and faster triplex PCR method as previously reported (16). This categorizes E. coli isolates into four major phylogenetic groups A, B1, B2, and D. The PAI usp (putative pathogenicity island carrying uropathogenic-specific protein) subtypes, Ia, Ib, IIa, and IIb were also determined according to their DNA sequence in usp variants, uspI and uspII (17,21). The present study aimed to investigate the prevalence of uropathogens among patients admitted to tertiary care hospitals in Islamabad, Pakistan, and to study the antimicrobial resistance patterns of ESBLs and non ESBLs isolates to different classes of antibiotics. Further analysis was done to understand the distribution and correlation among phylogenetic groups, PAIusp subtype, gyr A and parC genes in uropathogenic E. coli.

Isolation and identification of uropathogens
Urine samples were collected from patients with UTI or suspected UTI who visited OPD or were admitted to one of three different hospitals in Islamabad, Pakistan: Pakistan Institute of Medical Sciences (PIMS), the Government Services Hospital, and the Capital Development Authority (CDA) Hospital Islamabad. A total of 628 urine samples were examined and cultured on Cystine Lactose Electrolyte-Deficient (CLED) agar as per standard microbiological protocols.

Inclusion and exclusion criteria
Patients' urine cultures yielding a pure growth of ≥105 CFU/ml were considered significant and isolates were further processed for identification and susceptibility. However, the patient's urine yielding a count of ≤105 CFU/ml was considered insignificant and was excluded from the study (21). Patients' demographic data were obtained from computerized laboratory reports.

Phenotypic detection of ESBLs
Screening for ESBLs was performed by both doubledisc diffusion and Broth Microdilution Assays, as reported previously (24). Briefly, in the Disc Diffusion Assay BD Sensi-discs (Becton Dickinson, USA) were used with the following concentrations; cefotaxime (CTX) 30 µg; cefotaxime 30 µg plus 10 µg clavulanate (CA); ceftazidime (CAZ) 30 µg; and ceftazidime 30 µg plus 10 µg clavulanate (CA). A test was considered positive for ESBLs when the zone for cefotaxime (CTX) was 27 mm, and the zone for ceftazidime (CAZ) was 22 mm, and if the diameter of zones of inhibition was ≥5 mm increased in the zone of inhibition for the CAZ/CA and CTX/CA-containing discs versus the corresponding CAZ or CTX disc. Broth microdilution test was considered positive if MIC for either drug alone was 2 mg/l and if there was a decrease of at least 3 twofold dilutions for the combinations with clavulanate.

PCR amplification and sequencing
gyrA and parC genes were identified and amplified by PCR using primers described previously (25). PCR experiments were carried out using PTC-200 Peltier thermal cycler (SDS Diagnostics, Sweden) with primers from Sigma (Sigma Genosys Ltd, Sigma-Aldrich House, UK) according to the following conditions: denaturation at 95 °C for 5 min, 30 cycles of 94 °C for 15 sec, 53 °C for 20 sec, and extension at 72 °C for 1 min. PCR products were visualized by agarose gel electrophoresis. Purification was done before DNA sequencing using a QIA quick PCR purification kit (Qiagen, VWR International AB, Stockholm, Sweden) and quantified using a NO-1000 spectrophotometer (NanoDrop Technologies, USA). PCR products were sequenced at Macrogen Inc., Seoul, South Korea.

Phylogenetic grouping of uropathogenic E. coli
The phylogenetic group was determined by the triplex-PCR-based method reported previously (26). The PCR based method uses three primer pairs ChuA-1, ChuA-2, YjaA-1, YjaA-2 and TSPE4C2.1, TSPE4C2.2 to amplify the bacterial DNA, which generated fragment sizes of 279, 211, and 152 bp, respectively. E. coli isolates were classified into phylogenetic groups A, B1, B2, and D by using the results of PCR amplification ( Table 1). Subtypes of PAI usp were recorded by the PCR technique as reported previously (17). PAI usp variants were classified into four subtypes, Ia, Ib, IIa, and IIb according to the DNA sequence of the usp variants.
A total of 466 uropathogens including E. coli and K. pneumoniae were screened for their drug susceptibility patterns by the disc-diffusion method. Out of 334 isolates tested, E. coli exhibited the highest resistance against ampicillin (87%). However, a relatively lower bacterial resistance (51%) was observed against β-lactam/βlactamase-inhibitor combination such as amoxicillinclavulanic acid. Among cephalosporins, E. coli had a higher resistance (77%) against first-generation antibiotic cefazolin, while resistance against second and third-generation cephalosporins was recorded as 75% and 69%, respectively. In aminoglycosides, amikacin (35%) showed better antibacterial activity than gentamicin (50%). Bacterial resistance was also higher against quinolone and other tested drugs. In the case of K. pneumoniae, the highest rate of resistance was observed against ampicillin (100%) and the lowest against meropenem (21%). Resistant isolates were also in higher proportion against various tested drugs.

Discussion
In our study, the most commonly isolated organism in UTI was E. coli (53.2%) followed by K. pneumoniae (21%) and Enterobacter (11.6%). These results are per findings of previous studies (2,4,8,20,26). Furthermore, most of the UTIs were observed to be community-acquired (86%) and the frequency of E. coli was more than that of K. pneumoniae in both community and hospital-acquired infections. These results are supported by another study, in which UTIs accounted for about 63% of hospital-acquired infections and 86% were community-acquired (27). Our study also indicated a predominance of women over men (57% vs 43%), when gender-wise distribution was compared; a previous study (27), also reported a higher prevalence of UTIs among females than males (83% vs. 50 %) and E. coli was the most frequent uropathogen in both genders. E. coli caused 48.29% of urinary tract infections in females and 34.44% in males (9). Other studies also found that women were more likely to have UTIs than men in different geographical regions (28,29). Regarding the prevalence of UTIs by patients' gender, our findings are in good agreement with several reports from Pakistan and Bangladesh, which showed that these infections are more common in females as compared with males (6,9,30). The higher risk in women is mostly due to the shortness of the female urethra, which is 1.5 inches as compared with 8 inches in men. The prevalence of UTIs was found to be higher in older patients of both genders, whereas, it was low in younger patients having age <20 years. A similar trend has been reported earlier (31), in which UTIs among the elderly, adults, and children were 58.7%, 36.2%, and 5.1%, respectively. The possible causes of the higher incidence of UTIs in the elderly may be due to various factors including urinary tract anomalies, compromised immune response, malnutrition, functional disability, diabetes, and prostate enlargement in males and postmenopausal hormonal changes in females (2,32). Most commonly prescribed antibiotics to treat urinary tract infections are penicillins (amoxicillin-clavulanic acid), cephalosporins, carbapenems, fluoroquinolones (ciprofloxacin), co-trimoxazole, and aminoglycosides (33).
In the present study, the prevalence of ESBL producers was 65 % in the case of E. coli and K. pneumoniae similar to another study (18). In contrast, other research (19) has indicated a lower prevalence of ESBL-producing E. coli (9 %). Many other reports from different countries and regions showed different prevalence rates of ESBL producing Enterobacteriaceae causing urinary tract infections. In a previous study, the prevalence of ESBL producing multidrug-resistant uropathogens E. coli and Klebsiella sp. was 53% (34). Amoxicillin-clavulanic acid and cephalosporins are the common treatment against ESBL producing uropathogens in treatment centers (35). ESBL-producing E. coli and K. pneumoniae harbor many ESBL genes on large plasmids, these genes are not only responsible for the higher resistance levels to β-lactam antibiotics but also encode resistance to many other antibiotics including aminoglycosides, quinolones, chloramphenicol, sulfonamides, and tetracycline. ESBL is the presence of a particular beta-lactamase gene that produces an enzyme that is active on a wide spectrum of beta-lactam antibiotics (Amoxicillin, Cefotaxime, Aztreonam, etc.). Usually, the gene is on a plasmid together with many other resistance genes. ESBL works like all other beta-lactamases e.g., breaking the beta-lactam ring (36). Isolated uropathogens (E. coli and Klebsiella) in this study were highly resistant to ampicillin (87% vs 100%) and least resistant to meropenem (8% vs 21%), respectively. This is similar to the reports of various previous studies (18,37,38,39). However, the isolates exhibited less resistance to chloramphenicol (45% vs 32%), amikacin (35% vs 26%), and nitrofurantoin (25%vs 26%). Also, unexpected higher resistance was detected against amoxicillinclavulanic acid, ciprofloxacin, cephalosporins, trimethoprim and trimethoprim-sulfamethoxazole, and nalidixic acid. Self-medication, empirical therapy without culture sensitivity testing, and incomplete course of treatment in a community setting may be the major reasons for the development of drug resistance in underdeveloped countries like Pakistan (40). In our results of antimicrobial resistance, the profile is consistent with many previously reported studies (2,8,9,37) which indicated that Amikacin, imipenem, and meropenem were highly effective against Gramnegative bacilli causing UTIs and declared the highest resistance to penicillin followed by cephalosporins (first, second, and somewhat third-generation). However, resistance levels to these antimicrobial agents are high enough and clinicians are advised to change these agents in the treatment of urinary tract infections. High-level chloramphenicol resistance in K. pneumoniae and E. coli is caused by acquisition of the gene Cat (chloramphenicol acetyltransferase), but low-level resistance is caused by mutations that up-regulate drug efflux and mediated by cml and floR genes. (41). In this study, MIC levels of E. coli and K. pneumoniae showed a high degree of resistance against trimethoprim, followed by trimethoprim-sulfamethoxazole, ciprofloxacin, and amoxicillin-clavulanic acid. However, a relatively low resistance rate was found against chloramphenicol in

Conclusion
The findings of the present study are alarming in the context of increased resistance among uropathogens to most commonly prescribed antibiotics including penicillin, cephalosporins, and fluoroquinolones. The possible reason for this may be prolonged use of antimicrobial drugs, lax controls on drug availability, self-medication, and prescribing antibiotics without antibiotic susceptibility tests. Moreover, developing countries like Pakistan, Bangladesh, and India have a problem with overuse or misuse of antibiotic agents. Furthermore, there is a strong association between specific patterns of resistance and particular genotypes of the isolates but the relationship is complex. More intensive studies regarding the development of resistance within different phylogenetic groups could be used to predict the general basis for the development of antibiotic resistance. Monitoring, proper screening of multi-drug resistant isolates, and restricted use of antimicrobial agents is necessary and recommended to control high resistance rates of pathogens.